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Mannose and mannobiose specific responses of insect associated cellulolytic Streptomyces.

ABSTRACT: The cellulolytic insect symbiont bacterium, Streptomyces sp. SirexAA-E (SirexAA-E), is known to secrete a suite of Carbohydrate Active enZymes (CAZymes) for plant cell wall degradation in response to the available carbon sources. In this study, we sought to examine a poorly understood response of this bacterium to mannan, one of the major components of the plant cell wall, particularly in pine trees, the preferred host for SirexAA-E. SirexAA-E grew well on glucose, mannose, carboxymethyl cellulose (CMC), and locust bean gum (LBG) as sole carbon sources in the culture medium. The secreted proteins from each culture supernatant were tested for the polysaccharide-degrading ability, and the composition of secreted CAZymes in each culture supernatant were determined by LC-MS/MS. The results indicated that mannose, LBG, and CMC induced the secretion of mannan and cellulose-degrading enzymes. We found two α-1,2-mannosidases that were secreted during growth on mannose and LBG. By genomic analysis, we found a unique 12 bp palindromic sequence motif (5’-GACAACGTTGTC-3’) at 4 locations in the SirexAA-E genome, two of which were found upstream of genes encoding the above-mentioned α-1,2-mannosidases, along with a newly identified novel mannose and mannobiose responsive transcriptional regulator, SsManR. Furthermore, a transcriptional repressor that we previously showed was responsive to cellobiose, SsCebR, was determined to also use mannobiose as an effector ligand by in vitro binding assays. To test whether mannobiose induces the sets of genes under control of the two regulators, SsManR and SsCebR, SirexAA-E was grown on mannobiose, and the secretome composition was analyzed. As hypothesized, the composition of the mannobiose secretome combined sets of CAZymes found in both LBG and CMC secretomes, and so are likely under the regulation of both SsManR and SsCebR. To summarize, our findings support a transcriptional network that regulates mannose and LBG utilizing genes in SirexAA-E. Overall, this study defines a novel mannose regulatory transcriptional network that enables SirexAA-E to aggressively hydrolyze mannan-containing wood materials.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Streptomyces Sp. Sirexaa-e

SUBMITTER: Taichi E. Takasuka

LAB HEAD: Taichi E Takasuka

PROVIDER: PXD022202 | Pride | 2021-05-06

REPOSITORIES: Pride

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Project description:Brazil is the world’s second largest producer of ethanol. Increasing demand for this biofuel has called for investment in new technologies. One example of such technologies is the second-generation (2G) ethanol production. Lignocellulose comprises mainly cellulose and hemicellulose, which consist of high-energy sugars that can be converted into ethanol. Aspergillus sp play an important role in the recycling of lignocellulosic biomass. These species have been investigated as a cell factory to produce industrial enzymes. To improve our understanding of the mechanisms used by Aspergillus sp during degradation of plant biomass and to identify the hydrolytic enzymes secreted by these fungi, we have analyzed the transcriptome and secretome of an Aspergillus species grown on sugarcane bagasse (SEB). A. fumigatus was cultivated in the presence of fructose or SEB. Its cellulolytic and xylanolytic activities depended on time. The maximum activities of endoglucanase and xylanase from the fungus grown on SEB were 0.0029 and 10.82 U mL-1, respectively. Characterization of the transcriptome by RNAseq technology helped to identify genes that participated in the degradation of the biomass demonstrating potential application in the process of enzymatic hydrolysis. The RNAseq data revealed that 2287 genes were differentially expressed in the fungus grown on SEB; 1181 and 1046 of these genes were up- and down-regulated, respectively. There were several CAZymes among the up-regulated genes. Most of these enzymes belonged to the GH family, which includes important hydrolytic and accessory enzymes involved in the degradation of lignocellulose. Similarly, proteomic studies showed that a total of 130 proteins existed in the fungus grown on SEB. These proteins were classified into several groups of secreted extracellular enzymes, and their functional classification demonstrated that 59% of the proteins were CAZymes such as GH45-endoglucanases, GH6 and GH7-cellobiohydrolases, GH3-β-glucosidases, GH10-xylanases, and AA9-LPMO. Data obtained by transcriptome and secretome analyses indicated that A. fumigatus produces a considerable number of CAZymes that participate in the hydrolysis of biomass. These CAZymes are valuable for the lignocellulosic bioenergy industry, provide information for future studies on the degradation of biomass, and improve understanding of the role genes and enzymes play in the degradation process.

Project description:The search for new enzymes and microbial strains to degrade plant biomass is one of the most important strategies for improving the conversion processes in the production of environment-friendly chemicals and biofuels. In this study, we report a new Paenibacillus isolate, O199, which showed the highest efficiency for cellulose deconstruction in a screen of environmental isolates. Here, we provide a detailed description of the complex multi-component O199 enzymatic system involved in the degradation of lignocellulose. We examined the genome and the proteome of O199 grown on complex lignocellulose (wheat straw) and on microcrystalline cellulose. The genome contained 476 genes with domains assigned to carbohydrate-active enzyme (CAZyme) families, including 100 genes coding for glycosyl hydrolases (GHs) putatively involved in cellulose and hemicellulose degradation. Moreover, 31% of these CAZymes were expressed on cellulose and 29% on wheat straw. Proteomic analyses also revealed a complex and complete set of enzymes for deconstruction of cellulose (at least 22 proteins, including 4 endocellulases, 2 exocellulases, 2 cellobiohydrolases and 2 -glucosidases) and hemicellulose (at least 28 proteins, including 5 endoxylanases, 1 -xylosidase, 2 xyloglucanases, 2 endomannanases, 2 licheninases and 1 endo--1,3(4)-glucanase). Most of these proteins were secreted extracellularly and had numerous carbohydrate-binding domains (CBMs). In addition, O199 also secreted a high number of substrate-binding proteins (SBPs), including at least 42 proteins binding carbohydrates. Interestingly, both plant lignocellulose and crystalline cellulose triggered the production of a wide array of hydrolytic proteins, including cellulases, hemicellulases and other GHs. Our data provide an in-depth analysis of the complex and complete set of enzymes and accessory non-catalytic proteins—GHs, CBMs, transporters, and SBPs—implicated in the high cellulolytic capacity shown by this bacterial strain. The large diversity of hydrolytic enzymes and the extracellular secretion of most of them supports the use of Paenibacillus O199 as a candidate for second-generation technologies using paper or lignocellulosic agricultural wastes.

			Action	DRS
	5.SirexAA-E.fasta	Fasta
	5_SirexAA-E_LBG.msf	Msf
	5_SirexAA-E_LBG.mzData	Other
	5_SirexAA-E_cellulose.msf	Msf
	5_SirexAA-E_cellulose.mzData	Other

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Mannose and mannobiose specific responses of insect associated cellulolytic Streptomyces.

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